CN100494352C - Immunocolloidal gold test strip for detecting bovine tuberculosis antibody and preparation method thereof - Google Patents

Abstract

The invention discloses an immune colloidal gold indicator paper and making method to test cow tuberculosis antibody, which comprises the following parts: sample pad, connecting pad, cellulose nitrate film, absorbent pad and PVC backing liner, wherein the sample pad, connecting pad, cellulose nitrate film and absorbent pad are attached on the PVC backing liner; the connecting pad clads MPB83 protein-colloidal gold marker; the purified MPB70 protein is cladded on the cellulose nitrate film, which constitutes detecting line and quality control line formed by pure rabbit-anti-MPB83 protein lgG. The invention also provides the making method of cow mycobacterium astopic antigen MPB83, which possesses the recombinant Escherichia coli BL21/pET28a-MPB83 in the CCTCC with reserving number at CCTCC NO:M206142.

Description

Immunocolloidal gold test strip for detecting bovine tuberculosis antibody and preparation method thereof

technical field

The invention relates to the technical fields of animal bacteriology and animal infectious disease. Specifically relates to an immune colloidal gold test strip for detecting bovine tuberculosis antibody and a preparation method thereof.

Background technique

Bovine tuberculosis is a zoonotic infectious disease that seriously endangers the dairy industry and is caused by Mycobacterium bovis infection. It is classified as Class B zoonotic disease by the International Organization for Animal Health (OIE). Bovine tuberculosis costs world agriculture more than all other diseases of cattle combined, at more than $3 billion a year. Mycobacterium bovis can infect not only cattle and other mammals, but also humans. The etiological surveys of human tuberculosis at home and abroad have found that Mycobacterium bovis is one of the important causes of human tuberculosis. Therefore, the report of the Seventh Meeting of the World Health Organization Expert Committee pointed out: Unless bovine tuberculosis is eliminated, the control of human tuberculosis will not be successful (Wang Zhongren. The relationship between bovine tuberculosis and human tuberculosis. Chinese Journal of Antituberculosis, 2005, 27 (5): 123-125).

Since no effective drugs and vaccines have been found so far for the prevention and treatment of bovine tuberculosis, the eradication of bovine tuberculosis mainly adopts the international general policy of quarantine and culling positive cattle. Tuberculin (purified protein derivatives, PPD), also known as purified protein derivatives, is a metabolite produced by Mycobacterium tuberculosis when it grows in a liquid medium. It contains a variety of antigenic components and has a high sensitivity when used to detect tuberculosis . Therefore, tuberculin intradermal allergy is a legal quarantine method recognized by the International Organization for Animal Health and my country. However, since tuberculin may contain the same non-specific antigen components as other mycobacteria, false positives are prone to occur during detection; tuberculin intradermal allergy is not sensitive to open tuberculosis and systemic tuberculosis in the late stage of infection, while This stage is the dangerous time for TB patients to spread the disease. For the above reasons, the specificity of tuberculin intradermal allergy has been questioned. In addition, tuberculin intradermal allergy can only be tested in vivo, but cannot be retrospectively analyzed on preserved samples, and it is not suitable for the general survey of bovine tuberculosis in wild animals and remote mountainous areas.

Aiming at the defects of tuberculin intradermal allergy, the widely used diagnostic method for bovine tuberculosis at home and abroad, the animal pathogen division of the State Key Laboratory of Agricultural Microbiology of the applicant has developed a method for detecting bovine tuberculosis antibody by indirect hemagglutination method. kit (patent application number: 200510019996.1). In view of the technology at that time, the kit still has the following points to be improved. For example, only MPB70 single antigen is used in the kit, and the detection sensitivity needs to be further improved; the 1% sensitized sheep red blood cells used are stored at 4°C The period is only half a year.

Greenwald proved that MPB83 and MPB70 are the main specific secreted antigens of Mycobacterium bovis (Greenwald R, Esfandiari J, Lesellier S et al. Improved serodetection of Mycobacterium bovis infection in badgers (Meles meles) using multiantigen test formats. Diag Microbiol Infect Dis, 2003, 46: 197-203.). MPB83 is a bacterial surface-associated lipoprotein, which not only triggers cellular immunity but also humoral immunity in vivo, and has immunogenicity and immune protection (Feng C G, Palendira U C, Demangel J M, et al.Priming by DNA immunizationaugments protective efficacy of Mycobacterium bovis bacille calmette-guerin against tuberculosis [J]. Infect Immun, 2001, 69: 4174-4176.). MPB83 is an early secretion target protein (O′Loan CJ, Pollock JM, Hanna J, Neill SD. Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen[J]Lab.ClinImmunol, 1994, 1:608-611.), can be recognized by the human and animal immune system in the early stage of the disease, and thus has great significance for the early diagnosis of tuberculosis.

MPB70 is a specific protein secreted by Mycobacterium bovis, and its secretion accounts for 10% of the total protein in the virulent Mycobacterium bovis medium. It is an important humoral and cellular immune target antigen in the process of Mycobacterium bovis infection (Harboe M, Nagai S.MPB70, a unique antigen of Mycobacterium bovis BCG[J].Am Rev Resp Dis.1984, 129:444-452), is also one of the main components of PPD. The mature MPB70 protein in Mycobacterium bovis is very stable and is an ideal detection antigen (MarkD.Carr, Marieke J.Bloemink, Ellen Dentten et al.Solution Structure of the Mycobacterium tuberculosis Complex Protein MPB70[J].Biological Chemistry.2003, 278(44):43736-43743.). MPB83 is a homologous protein with 61% identity to MPB70 (Nagai S, Matsumoto J, Nagasuga T. Specific skin-reactive protein from culture filter of Mycobacterium bovis BCG [J]. InfectImmun.1981, 31(3): 1152-1160. ), our laboratory has proved that MPB70 can react with the antiserum of MPB83 by enzyme-linked immunosorbent assay (ELISA) and agar diffusion method, and MPB83 can also react with the antiserum of MPB70, which shows that MPB70 and MPB83 have a common antigen Determined family.

Colloidal gold immunochromatography (gold-immunochromatography assay GICA) is a new immunoassay method developed in the 1980s. It is a method of applying colloidal gold labeling technology and colloidal gold as a tracer to antigen-antibody reactions. A novel immunolabeling technique. It has the advantages of simplicity, rapidity, strong specificity, high sensitivity and low cost.

At present, the application of colloidal gold immunochromatography technology to the in vitro detection of bovine tuberculosis antibody is still blank in China. The only commercialized kit abroad is the "Anigen Rapid Bovine TB Ab TestKit" produced by Animal Genetics, Inc. in Korea. The kit is Using MPB70 protein as the detection antigen has good specificity, but the sensitivity is not high, and it may be missed in clinical practice. The "Immune Colloidal Gold Test Strip for Detecting Bovine Tuberculosis Antibodies" developed by the applicant is the first case at home and abroad that simultaneously uses two proteins, MPB70 and MPB83, as detection antigens, and has high specificity and high sensitivity.

Contents of the invention

The object of the invention is to overcome the deficiencies in the prior art, provide a kind of specificity strong, high sensitivity, the immune colloidal gold test strip of the antibody of the detection bovine tuberculosis that antigenic protein is convenient to store and carry and preparation method thereof, for the prevention and control of bovine tuberculosis in my country The system provides a simple detection and diagnosis method and tool.

The general technical roadmap of the present invention is as shown in accompanying drawing 1.

The present invention is realized through the following technical solutions:

In order to realize the present invention, the applicant constructed a recombinant Escherichia coli BL21/pET28a-mpb83 capable of secreting Mycobacterium bovis antigen MPB83, which was preserved in the China Center for Type Culture Collection on December 21, 2006 ( CCTCC), its preservation number is CCTCC NO: M206142. The MPB83 secreted by the recombinant strain was used as the colloidal gold-labeled antigen.

Another recombinant Escherichia coli BL21/pET28a-mpb70 used in the present invention that can secrete Mycobacterium bovis antigen MPB70 has a preservation number of CCTCC NO: M205135, and the MPB70 secreted by the recombinant strain is used as the coating antigen of the detection line.

As shown in accompanying drawing 2, the immune colloidal gold test strip of detection bovine tuberculosis antibody of the present invention, it comprises sample pad (1), binding pad (2), nitrocellulose membrane (3), water-absorbing pad (4) and The PVC backing (7), its specific structure is: on the PVC backing (7), there are sample pads (1), bonding pads (2), nitrocellulose membranes (3), water-absorbing pads (4 ); the described binding pad (2) is coated with MPB83 protein-colloidal gold marker; the described nitrocellulose membrane (3) is coated with detection line (5) and A quality control line (6) composed of purified rabbit anti-MPB83 protein IgG. All the above materials except antigen and antibody were purchased from Millipore Company.

The method for preparing the above-mentioned immune colloidal gold test strip for detecting bovine tuberculosis antibody, its steps include:

1) Cloning Mycobacterium bovis-specific secretory protein MPB83, transforming Escherichia coli, obtaining and expressing recombinant Escherichia coli with the preservation number CCTCCNO: M206142;

2) Purifying the MPB83 protein described in step 1) and the MPB70 protein with the preservation number CCTCC NO: M205135;

3) immunizing healthy rabbits with the MPB83 protein prepared in step 2) to obtain rabbit anti-MPB83 IgG antibodies;

4) Colloidal gold is prepared by reacting trisodium citrate and chloroauric acid;

5) adding the MPB83 protein prepared in step 2) to the colloidal gold prepared in step 4) to obtain the MPB83 protein-colloidal gold marker;

6) Coating the MPB83 protein-colloidal gold marker prepared in step 5) on the binding pad (2);

7) coating the MPB70 protein prepared in step 2) on a nitrocellulose membrane (3) to form a detection line (5); and coating the IgG of the rabbit anti-MPB83 protein prepared in step 2) on a nitrocellulose membrane (3) ) constitutes the quality control line (6);

8) Adhering the sample pad (1), binding pad (2), nitrocellulose membrane (3) and water-absorbing pad (4) in sequence on the PVC backing (7) to obtain the Immunocolloidal gold test strips for the detection of bovine tuberculosis antibodies.

The invention clones the coding gene of the Mycobacterium bovis specific antigen MPB83, constructs the prokaryotic expression carrier, makes it expressed in Escherichia coli, and purifies the MPB83 protein for future use. At the same time, the MPB70 protein was also purified for future use. Select the purified MPB70 as the coated antigen of the detection line, use the purified MPB83 as the colloidal gold-labeled antigen, use the double antigen sandwich to detect the corresponding antibody in the test sample (serum), and judge according to the color depth or presence of the detection line Whether the sample liquid to be tested contains antibodies to MPB83 and MPB70 antigens. If there is no red band on the quality control line, the test strip is invalid (see the examples for specific usage methods).

Compared with the existing domestic and foreign bovine tuberculosis antibody detection kits, the present invention has the following outstanding advantages:

1. The immunocolloidal gold test strip for detecting bovine tuberculosis antibody of the present invention uses two antigenic proteins secreted by Mycobacterium bovis, namely MPB83 and MPB70, so it has both high specificity and high sensitivity.

2. The detection test strip of the present invention has a short detection time (20 minutes) and does not require any special instruments and equipment, and the detection cost is low.

3. The detection test strip of the present invention is easy to operate and does not need to be operated by professionals.

4. The detection test strip of the present invention is convenient to store and does not require high temperature. The effective storage period can reach up to two years at 4-8° C.; it can be stored at room temperature for 12 months.

Description of drawings

Figure 1: Overall technical roadmap of the present invention

Figure 2: Schematic diagram of the assembly of the detection test strip of the present invention

In the figure: 1 is the sample pad, 2 is the binding pad, 3 is the nitrocellulose membrane, 4 is the absorption pad, 5 is the detection line, 6 is the quality control line, 7 is the PVC backing

Figure 3: Schematic diagram of judging the results of the test strips of the present invention

In the figure: A: the result of positive standard product, B: the result of positive sample, C: the result of negative sample, D, E: the failure of the test strip.

Figure 4: is the physical diagram of the vector construction of the present invention.

Figure 5: PCR amplification results of mpb83 gene.

In the figure: 1, 2, 3: the amplified target gene; M: the molecular weight standard of DL2000

Figure 6: Identification results of the recombinant plasmid pET28a-mpb83

In the figure: M1: molecular weight standard of DL15000 (from top to bottom are 15000bp, 10000bp, 7500bp, 5000bp, 2500bp, 1000bp, 250bp); M2: molecular weight standard of DL2000 (from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp); p: BL21/pET28a(+); rp: BL21/pET28a-mpb83

Note: Nos. 1-4 are transformants from different single colonies; No. 2 is the recombinant plasmid identified correctly after digestion Fig. 7 shows MPB83 protein expression situation under different IPTG induction concentration and induction time

Figure 8: SDS polyacrylamide gel denaturation electrophoresis of purified MPB83 protein

In the figure: M: protein molecular weight standard; 1, 2: purified MPB83 protein

Figure 9: Western blot analysis of purified MPB83 protein

In the figure: M: Protein molecular weight standard; 1: Purified MPB83 protein and bovine tuberculosis negative serum; 2: Purified MPB83 protein and bovine tuberculosis positive serum

Figure 10: SDS polyacrylamide gel denaturation electrophoresis pattern of purified rabbit anti-MPB83 protein IgG

In the figure: M: protein molecular weight standard; 1, 2: purified MPB83 protein IgG

Detailed ways

Embodiment 1 (preparation embodiment)

(1) Cloning of the Mycobacterium bovis-specific secretory protein MPB83 gene and its expression and purification in recombinant Escherichia coli 1 Cloning of the cDNA sequence of the Mycobacterium bovis-specific antigen gene mpb83

(1) PCR primer design and synthesis

The upstream and downstream primers were designed according to the mpb83 gene sequence in Genbank (accession number: GI: 31794050). The upstream primer introduced an EcoRI restriction site (as indicated by the underline in the primer), and designed 6 protective bases, and the downstream primer introduced a HindIII restriction site (as indicated by the underline in the primer), and designed 6 protective bases. The primers of the present invention were synthesized by Beijing Aoke Biological Company.

mpb83 upstream primer: 5'-AGTTCA GAATTC ATGATCAACGTTCAGGCCAAAC-3'

mpb83 downstream primer: 5'-ACGTCG AAGCTT TTACTGTGCCGGGGGCATCA-3'

(2) MPB83 protein coding gene amplification and processing

The standard strain of Mycobacterium bovis ((Mycobacterium bovis AF2122/97, the strain was donated by researcher Guo Mingxing of Hubei Entry-Exit Inspection and Quarantine Bureau, its gene sequence has been registered in Genebank, accession number: NC002945) bacterial culture plus a small amount of double Boil in distilled water for 10min to lyse the bacteria, centrifuge and take the supernatant as a PCR template. The PCR amplification reaction system is: 10×Taq Buffer 5.0μl, 25mmol/LMgCl ₂ 1.0μl, 2mmol/L dNTPs 1.5μl, 20μmol/L 1.0 μl downstream primers, 1.0 μl TaqDNA polymerase, 3 μl template, and add sterile double distilled water to 50 μl. PCR reaction conditions: denature at 94°C for 4 min, enter 30 cycles (denaturation at 94°C for 30 sec, renaturation at 60 for 30 sec, 72 ℃ extension 2min), and finally 72 ℃ extension 10min. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and a mpb83 gene fragment (as accompanying drawing 5) of size 660bp. Use DNA gel fast recovery kit (purchase The mpb83 gene fragment was purified from Shanghai Bioengineering Company.

2 Construction of prokaryotic expression vector of Mycobacterium bovis specific antigen gene mpb83

(1) Construction of recombinant Escherichia coli DH5α/pET28a-mpb83

The PCR product was recovered, purified, connected to the cloning vector pMD18-T (a commercial plasmid vector, purchased from Dalian Bao Biological Co., Ltd.), transformed into Escherichia coli DH5α, screened positive clones, extracted a small amount of plasmid, and used restriction endonuclease The recombinant plasmid was identified by enzyme digestion, and its size was confirmed to be in line with the expectation by 0.8% agarose gel electrophoresis; it was confirmed by sequencing that the fragment had no base mismatch.

pET28a(+) (purchased from Invitrogen) and PCR product were digested with EcoRI and HindIII, purified and recovered with DNA Gel Quick Extraction Kit (purchased from Shanghai Bioengineering Co., Ltd.), and then ligated with T41igase for sticky end ligation, 16 ℃ water bath overnight, transform DH5α competent bacteria, culture at 37 ℃, pick bacteria, extract recombinant plasmid, and identify by enzyme digestion (see Figure 6). The recombinant expression plasmid was named pET28a-mpb83.

3 Construction of recombinant Escherichia coli Escherichia coli BL21/pET28a-mpb83 (as accompanying drawing 4)

Transform the recombinant expression vector pET28a-mpb83 with the correct sequence identification into Escherichia coli BL21 (the Escherichia coli strain was purchased from Stratagene Company) competent cells, coat LB kanamycin (to a final concentration of 50 μg/mL) plate, and select the plate A single positive colony (BL21/pET28a-mpb83) was placed in LB liquid medium and cultured with shaking at 37°C until the logarithmic growth phase (OD ₆₀₀ =0.6-0.8), and isopropylthio-β-D was added to the medium -Galactoside (IPTG) induced expression, followed by SDS polyacrylamide gel denaturing electrophoresis and Western-blot detection (Sambrook J, Fritsch EF, Manny Artis T editors, Molecular Cloning Experiment Guide, Jin Dongyan , Li Mengfeng and other translations, second edition, Science Press, Beijing, 1992 edition), and simultaneously transform the original vector pET28a into Escherichia coli BL21 (DE3) cells as a blank control bacterium (BL21/pET-28a).

3 Prokaryotic Induced Expression of Mycobacterium bovis Specific Antigen Protein MPB83

(1) Determination of optimal inducer concentration and optimal induction time

Pick a single colony of recombinant E. coli Escherichia coli BL21/pET28a-mpb83 into 5 mL LB medium, add kanamycin to a final concentration of 50 μg/mL, culture on a shaker at 37°C for 8 hours, and then put it in a refrigerator at 4°C overnight. After diluting the bacterial solution with LB medium containing 50 μg/mL kanamycin at a ratio of 1:100, divide it into 2 bacterial culture bottles (10 mL/bottle), and culture it on a shaker at 37 ° C until logarithmic growth period (OD600=0.6-0.8), add the inducer isopropylthio-β-D-galactoside (IPTG) to the final concentration of 0.4, 0.8mol/L, respectively, after induction 0, 1, 2 3. Take 1mL of bacterial culture at 3 and 4 hours, centrifuge at 12000r/min to collect the bacteria, rinse the pellet with PBS and resuspend in 50μL, add 40μL of 2×SDS sample buffer and 10uL of DTT, boil at 100°C for 10min, take 20μl for SDS polypropylene Amide gel denaturing electrophoresis analysis (as shown in Figure 7) (Edited by Sambrook J, Fritsch EF, Maniartis T, "Molecular Cloning Experiment Guide", the second edition, Beijing Science Press, 1992 edition) . According to the expression of MPB83 protein, the optimal induction concentration of IPTG was determined to be 0.8mmol/L, and the optimal induction time was 3 hours.

(2) Mass-induced expression and protein purification of Mycobacterium bovis-specific secretory protein MPB83

Pick a single colony from the plate to 5 mL LB medium, add Kanamycin (Kan) to a final concentration of 50 μg/mL, culture on a shaker at 37°C until turbid (about 8 hours), and put it in a refrigerator at 4°C overnight. Take 2 mL of the bacterial solution and add it to 200 mL LB/Kan medium, culture it on a shaker at 37 ° C for about 3 h to OD600 = 0.6-0.8, add the inducer IPTG to a final concentration of 0.8 mmol/L, and continue the induction culture for 3 h.

The induced bacterial cells were centrifuged and ultrasonically crushed to extract inclusion bodies, and after purification, denaturation, renaturation and dialysis, they were subpackaged and stored at -20°C for future use. With reference to Sam Brook J, Fritsch EF, Manny Artis T chief editor, "Molecular Cloning Experiment Guide", the second edition, Beijing Science Press, the method reported in 1992 edition to the purified protein (as shown in Figure 8 shown) for western blot analysis, the results are shown in Figure 9, confirming that the recombinant Escherichia coli can specifically express the Mycobacterium bovis-specific secretory protein MPB83, and the expressed protein exists in Escherichia coli BL21 in the form of inclusion bodies, and has immune learning activity.

(2) Preparation and purification of rabbit anti-MPB83 protein IgG

The purified MPB83 protein was immunized into healthy rabbits to prepare highly specific and high-titer rabbit anti-MPB83 protein hyperimmune serum, and the rabbit serum containing MPB83 antibody was centrifuged at 4000r/min for 10 minutes, and the supernatant was purified according to the following steps: Take 10ml of the supernatant Clear at 4°C, centrifuge at 12,000r/min for 10 minutes, discard impurities, then add 40ml of 0.06M pH4.5 acetate buffer, then slowly add 330μl octanoic acid at room temperature (25°C), and stir while adding dropwise. Slowly add saturated ammonium sulfate to a final concentration of 45%, dissolve with 0.01M PBS (pH7.4) and dialyze overnight. SDS polyacrylamide gel denaturing electrophoresis analysis (as shown in Figure 10) showed two protein bands, one for the IgG heavy chain and one for the IgG light chain. The optical absorption value (OD) of the purified polyclonal antibody at 280nm and 260nm wavelength was measured with an ultraviolet spectrophotometer, and the polyclonal antibody concentration was calculated according to the formula 1.45×OD280-0.74×OD260. Adjust the volume of the solution so that the final concentration is 10 mg/ml. Use this antibody to coat the quality control line on the nitrocellulose membrane.

(3) Cloning of Mycobacterium bovis-specific secretory protein MPB70 gene and its expression and purification in recombinant Escherichia coli

For the cloning of Mycobacterium bovis-specific secretory protein MPB70 gene and its expression and purification method steps in recombinant Escherichia coli, please refer to the patent application number 200510019996.1, and the title of the invention is "a kit suitable for bovine tuberculosis antibody detection and Application" published content.

Embodiment 2 (preparation embodiment)

Mycobacterium bovis-specific secreted protein MPB83 and MPB70 antibody diagnostic kit assembly and preparation method 1. Kit assembly:

The kit of the present invention consists of the following: sample pad (1), binding pad (2), nitrocellulose membrane (3), water-absorbing pad (4) and PVC backing (7), and the specific structure and assembly sequence are: A sample pad (1), a binding pad (2), a nitrocellulose membrane (3), and an absorbent pad (4) are adhered to the backing (7) in sequence; the binding pad (2) is coated with MPB83 protein-colloidal gold marker; said nitrocellulose membrane (3) is respectively coated with a detection line (5) made of purified MPB70 protein and a quality control line (6) made of purified rabbit anti-MPB83 protein IgG .

2 Preparation of colloidal gold:

Dilute 1% chloroauric acid to 0.01% with ultrapure water, put it on a magnetic heating stirrer, stir and boil, add 2.0ml 1% trisodium citrate for every 100ml 0.01% chloroauric acid, and continue to boil until the liquid is orange-red That is, the heating was stopped, and the water loss was replenished after cooling to room temperature. The appearance of the prepared colloidal gold should be pure, translucent, free of sediment and floating matter, and the validity period is one year.

3MPB83 protein-colloidal gold marker preparation:

Under magnetic stirring, use 0.1M potassium carbonate to adjust the pH value of the colloidal gold to 6.5, add 2 μL 500 μg/mL MPB83 protein per ml of colloidal gold, continue to stir and mix for 30 minutes, add 10% BSA to a final concentration of 1%, and let it stand for 30 minutes . Centrifuge at 12000rpm and 4°C for 30min, discard the supernatant, and wash the precipitate with 0.02M borate buffer solution with pH9.0 (recipe: boric acid 0.1237g, PEG-200001g, dilute to 1000ml with ultrapure water, adjust pH to 9.0) Twice, use 0.02MpH9.0 borate buffer (recipe: boric acid 0.1237g, PEG-200001g, set the volume to 1000ml with ultrapure water, adjust pH to 9.0) with 0.02MpH9.0 borate buffer solution of 1/20 initial colloidal gold volume The precipitate was resuspended and stored at 4°C for use, with a validity period of 60 days.

4 Coating of binding pads

Soak the binding pad in 0.01M pH 7.4 phosphate buffer (recipe and preparation: 20g BSA, 25g sucrose, 3g polyvinylpyrrolidone (PVP-10), 0.2gNaN ₃ NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 12H ₂ O 2.9g, KH ₂ PO ₄ 0.2g, dilute to 1000ml with ultrapure water) for 30min, then dry at 37°C. Then, the prepared MPB83 protein-colloidal gold marker was evenly coated on the conjugation pad with a Biodot film spotting instrument, and 9 μl of MPB83 protein-colloidal gold marker was coated per cm of the conjugation pad, vacuum-dried, vacuum-packed, and placed at 4°C for use.

5 Handling of the sample pad

Soak the sample pad in 0.01M pH 7.4 phosphate buffer (recipe and preparation: 20g bovine serum albumin (BSA), 25g sucrose, 3gPVP-10, 0.2gNaN ₃ , NaCl 8g, KCl 0.2g, Na ₂ HPO ₄ 12H ₂ O 2.9g, KH ₂ PO ₄ 0.2g, dilute to 1000ml with ultra-pure water for 30min, dry at 37°C, vacuum seal, store at 4°C for later use.

6 Coating of nitrocellulose membrane

Coat the purified Mycobacterium bovis-specific secreted protein MPB70 (concentration: 3 mg/mL) on a nitrocellulose membrane with a Biodot film spotting instrument as a detection line, the coating volume is 0.6 μl/cm, and the detection line is close to the binding pad It is about 8mm away from the end of the binding pad; the purified rabbit IgG antibody against MPB83 protein (concentration 2mg/ml) was coated on the nitrocellulose membrane as a quality control line with a Biodot spotting membrane instrument, and the coating volume was 0.6 μl/ml. cm, the quality control line is close to the absorbent pad, about 8mm away from the absorbent pad, and the distance between the two lines is 5-8mm. Dry at 37°C and package for later use.

7 Assembly of test strips

Adhere the sample pad (1), binding pad (2), nitrocellulose membrane (3), and water-absorbent pad (4) to the PVC backing (7) in the order shown in Figure 2, and cut them into 3mm-wide strips. Strips, vacuum encapsulated. Stored at 4-8°C, the validity period is 2 years; stored at room temperature, the validity period is 12 months.

Embodiment 3 (application embodiment)

How to use the immune colloidal gold test strip for bovine tuberculosis antibody

1. Pretreatment of serum samples

The bovine serum samples were centrifuged at 4000rpm/min for 10min to remove blood cells, stored at 20°C for a long time and at 4°C for a short term.

2. Detection steps

Use a pipette to draw 150 μl of the serum to be tested and slowly drop it on the sample hole of the colloidal gold test strip, and observe the result after 20 minutes.

3. Result judgment

As shown in Figure 3, if the color of the detection line of the test strip of the sample to be tested is obviously darker than the color of the detection line of the test strip of the positive standard product, and a red band appears on the quality control line, it is judged as a positive sample, that is, there is Antibodies of Mycobacterium tuberculosis-specific secretory proteins MPB83 and MPB70 indicate that the cattle are tuberculosis cattle; if no color appears on the test strip detection line of the sample to be tested, and a red band appears on the quality control line at the same time, it is judged as a negative sample. That is, there is no antibody to the specific secretion protein MPB83 and MPB70 of Mycobacterium bovis in the sample to be tested, indicating that the cattle are tuberculosis-negative cattle; If there is no red band on the quality control line, the test strip is invalid.

Embodiment 4 (application embodiment)

Examples of application effects of the present invention

Refer to the operation steps described in Example 3 for the immunocolloidal gold test paper detection method for detecting bovine tuberculosis antibodies referred in this embodiment, and its detection results are shown in Table 1, Table 2 and Table 3.

1. Specificity test

Carry out test by the method described in embodiment 3, detect bovine tuberculosis positive and bovine tuberculosis negative serum (preserved by this laboratory, through PPD skin test, ELISA, pathology and bacteriology test), paratuberculosis bovine serum, brucellosis bovine serum, arcuate Insect bovine serum and foot-and-mouth disease bovine serum (both purchased from China Veterinary Supervision Institute). The test results (see Table 1) showed that no color appeared on the test lines of paratuberculosis bovine serum, brucellosis bovine serum, toxoplasmosis bovine serum and foot-and-mouth disease bovine serum samples, while red bands appeared on the quality control line. The test strip of the present invention has no cross-reaction with other diseases of cattle, showing that the test strip of the present invention has good specificity.

Table 1 The specificity test of test strip of the present invention

Note: "+" means positive, "—" means negative.

2. Sensitivity test

Carry out test by the method described in embodiment 3, detect with the test strip that the present invention develops after 10 parts of bovine tuberculosis positive serum doubling dilutions, and the reagent that detects bovine tuberculosis antibody with the indirect hemagglutination that the applicant has developed before Kit (the kit of 200510019996.1 patent application) was compared with similar Korean test strips (results are shown in Table 2). The comparison results show that the sensitivity of the test strip developed by the present invention is obviously higher than that of the test kit (indirect hemagglutination) of the 200510019996.1 patent application and similar Korean test strips.

Table 2 The sensitivity test results of test strips of the present invention and the applicant's previous patent application and Korean similar test strips

Note: The numbers in row 1 in Table 2 indicate the number of positive sera to be tested from 1 to 10, and the numbers in rows 2-4 in the table indicate the highest dilution factor that can detect positive.

3. Comparative test with tuberculin intradermal allergy (TST)

Tested according to the method described in Example 3, 61 clinical bovine serum samples were detected with bovine tuberculosis antibody detection test strips developed by the present invention, and compared with the TST method (the results are shown in Table 3). The comparison results showed that: 9 TST positive cattle, of which 3 were negative by the test strip test, and the positive coincidence rate was 66.66%. Of the 52 TST-negative cattle, 43 of them were judged to be negative by test strips, and the negative coincidence rate was 82.69%. The total coincidence rate is 80.33%.

Table 3 Comparison of test strips of the present invention and TST method

4. Repeatability test

Test according to the method described in Example 3, 10 parts of positive and 10 parts of negative samples selected use the test strips developed by the present invention of 5 different batches (061201, 061202, 061203, 061204, 061205) to detect, the result Show that this test strip has better repeatability, coefficient of variation <10% (results are shown in Table 4-1 and 4-2).

The test kit of the present invention of different batches of table 4-1 detects the result of 8 known positive samples

The kit of the present invention of table 4-2 different batches detects 8 known negative sample results

5. Shelf life test

(1) Destruction to test strip of the present invention under high temperature conditions

5 batches (batch numbers are 060815, 060930, 061020, 061112, 061201) test strips developed by the present invention were stored at 37°C for 6 days, then tested according to the method described in Example 3, and the same batch of test strips stored at 4°C The test strip simultaneously detected 10 bovine tuberculosis negative sera and 10 bovine tuberculosis positive sera. The result shows: 37 ℃ of action 6 days have no damage to the test strip of the present invention (as shown in table 5-1 and 5-2)

Table 5-1 High temperature (37 ℃ action 6 days) to the destructive action of test strip of the present invention (positive serum detection result)

Table 5-2 The destructive effect of high temperature (37°C for 6 days) on the test strip of the present invention (the detection result of negative serum)

(2) Storage period test at room temperature

The test strips of the present invention prepared in the same batch were stored at 4°C and room temperature for 1 to 12 months respectively, and were taken out at 0 months, 3 months, 6 months, 9 months and 12 months, according to the examples 3. The method described in 3 was tested, and 1 part of positive serum with multiple dilution was detected to carry out the storage period sensitivity test, and 10 known positive and negative sera were detected to carry out the storage period specificity test, and the sensitivity, specificity and stability were observed. , to determine the shelf life of the test strips. The results show that: the test strip of the present invention has no change in its specificity and sensitivity after being stored at room temperature for more than 12 months (the test results are shown in 5-3).

Test strip sensitivity test result of the present invention in table 5-3 different shelf life

Test strip specificity test result of the present invention in table 5-4 different shelf life

6. Detection of clinical samples

The test was carried out according to the method described in Example 3, and 1004 bovine sera from different test areas were tested, and the results are shown in Table 6. The positive rate of detected samples was 5.26%-70.5%. Clinical application proves that the kit of the present invention has good accuracy, repeatability, sensitivity and specificity, is suitable for clinical large-scale serum antibody detection and disease diagnosis, and has important significance in the clinical promotion and application of clearing the source of bovine tuberculosis infection and controlling bovine tuberculosis .

Table 6 The clinical application of test strips of the present invention

Although the content of the present invention is described in conjunction with this embodiment, it should not be regarded as limiting the scope of the present invention, which is defined by the appended claims. In addition, those skilled in the art may make various changes or modifications to the present invention within the scope defined by the appended claims, and these changes or modifications also fall within the protection scope of the present invention.

Claims (3)

1, comprising by preserving number is the bovine tuberculosis antibody immunity colloidal gold test paper strip of secreted mycobacterium tuberculosis var bovis antigen protein MPB83 of recombination bacillus coli (Escherichia coli) BL21/pET28a-mpb83 of CCTCC M206142 and reorganization mycobacterium tuberculosis var bovis antigen protein MPB70.

2, the described bovine tuberculosis antibody immunity colloidal gold test paper strip of claim 1, it comprises sample pad (1), pad (2), nitrocellulose filter (3), absorbent pad (4) and PVC backing (7), it is characterized in that, on PVC backing (7), be stained with sample pad (1), pad (2), nitrocellulose filter (3), absorbent pad (4) in order successively; Be coated with MPB83 albumen-colloid gold label thing on the described pad (2); Be coated with the nature controlling line (6) of the proteic IgG formation of the anti-MPB83 of rabbit of detection line (5) that the MPB70 albumen of purifying constitutes and purifying on the described nitrocellulose filter (3) respectively.

3, the preparation method of claim 1 or 2 described bovine tuberculosis antibody immunity colloidal gold test paper strips, its step comprises:

1) difference clened cows type mycobacteria specific secretory protein MPB83 and MPB70, transformed into escherichia coli obtains corresponding recombination bacillus coli;

2) prepare mycobacterium tuberculosis var bovis antigen protein MPB83 and MPB70 respectively by the described recombination bacillus coli of step 1);

3) to step 2) described antigen protein carries out purifying;

4) with step 2) preparation MPB83 albumen obtain the anti-MPB83IgG antibody of rabbit;

5) with trisodium citrate and hydrochloro-auric acid prepared in reaction Radioactive colloidal gold;

6) the antigen protein MPB83 with the step 3) preparation adds in the Radioactive colloidal gold of step 5), obtains MPB83 albumen-colloid gold label thing;

7) the MPB83 albumen-colloid gold label thing with the step 6) preparation is coated on the pad (2);

8) the MPB70 albumen with the step 3) preparation is coated on upward formation detection line (5) of nitrocellulose filter (3); And the anti-MPB83 IgG of rabbit of step 4) preparation is coated on nitrocellulose filter (3) goes up and constitute nature controlling line (6);

9) on described PVC backing (7), adhere to described sample pad (1), pad (2), nitrocellulose filter (3), absorbent pad (4) in order successively, obtain the immunity colloidal gold test paper strip of described detection bovine tuberculosis antibody.

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